Sexual attraction pheromone of tribolium castaneum and insect control device

ABSTRACT

A sexual attraction pheromone exclusively obtained from Tribolium castaneum male insects, a method to obtain said pheromone and a device useful for controlling insects in grain silos are disclosed. The device comprises an outer shell, a membrane impregnated with the sexual attraction pheromone and a nucleus containing an insecticide formulation comprising effective amount of an isolated microsclerotia of Beauvaria bassiana and an agronomically acceptable carrier.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority of U.S. Provisional Application No.62/815,536 filed on Mar. 8, 2019 under 35 U.S.C. 119(e), the entirecontents of which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to an insect sexual attraction pheromoneand to a device useful for controlling pests, in particular insects, ingrain silos. The device comprises an outer shell, a membrane impregnatedwith a sexual attraction pheromone extracted from Tribolium castaneummales and a nucleus comprising insecticide formulation.

BACKGROUND

Insect control devices with a “lure and trap” mechanism often include aluring composition such as an aggregation pheromone to attract insects,as well as an insecticide formulation or other pest control compounds.Several of these compounds may require carefully controlled conditionsin order to activate the biological mechanisms involved in theinsecticidal effect. For example, bioinsecticides based on dormant formsof several entomopathogenic fungi may be activated under adequatehumidity conditions. These conditions may be obtained through theaggregation of a certain number of insects in the vicinity of thesedormant forms. When the adequate conditions are met, the dormant formsactivate, and the insecticidal effect is achieved. A sexual attractionpheromone, which targets only insects of a certain sex, can beadvantageously used to achieve these controlled conditions.

4,8-dimethyldecanal, also known as tribolure, is an aggregationpheromone of Tribolium castaneum. Although it has been mistakenlyreported as a “sexual pheromone”, it has been found that this moleculeattracts both male and female insects, therefore not constituting a truesexual pheromone.

There is therefore a need to provide a sexual pheromone of Triboliumcastaneum useful for insect control devices employing a luringcomposition.

SUMMARY OF THE INVENTION

In a first aspect, an object of the present invention is an isolatedsexual attraction pheromone of Tribolium castaneum that is exclusivelyobtained from male insects. The pheromone has an average molecularweight of approximately 210 g/mol, a molecular formula of C₁₄H₂₆O and achemical structure having only one degree of unsaturation and a terminalcarbonyl group.

In a second aspect, an object of the present invention is a method toobtain an isolated sexual attraction pheromone from a plurality of maleinsects, comprising the steps of:

-   -   placing the plurality of male insects in an airtight container        comprising a solid support,    -   placing and maintaining the container on a stove to generate        volatile organic compounds,    -   separating the plurality of male insects from the solid support,        extracting the volatile organic compounds from the solid support        using solid-phase microextraction fibers, and    -   isolating of the pheromone from the volatile organic compounds        by capillary gas chromatography.

In a preferred embodiment of the second aspect, said solid support isselected from filter paper and a feed medium comprising flour.Preferably, the feed medium comprises about 30 g of flour.

In another preferred embodiment of the second aspect, the container ismaintained on a stove for a period of approximately one month.

In a third aspect, it as an object of the present invention an insectcontrol device comprising:

-   -   an outer shell comprising a plurality of orifices,    -   a central membrane impregnated with a luring composition, and    -   an active nucleus comprising an insecticide formulation.

In a fourth aspect, it as an object of the present invention an insectcontrol device comprising:

-   -   an outer shell comprising a plurality of orifices,    -   a central membrane impregnated with the isolated sexual        attraction pheromone of Tribolium castaneum of the first aspect        of the invention, and    -   an active nucleus comprising an insecticide formulation.

In preferred embodiments of the third and fourth aspects, said membraneis comprised in a capsule attached to the outer shell.

In preferred embodiments of the third and fourth aspects, said capsulefurther comprises a conical surface. Preferably, said conical surfacehas a smooth, polished and shining surface.

In further preferred embodiments of the third and fourth aspects, theinsecticide formulation comprises an effective amount of isolatedmicrosclerotia of Beauvaria bassiana and an agronomically acceptablecarrier. Such microsclerotia are described in U.S. patent applicationNo. 62/815,541, the contents of which are incorporated by referenceherein.

In yet further preferred embodiments of the third and fourth aspects,the active nucleus is located in a lower portion of the device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chromatograph with the main components of male Triboliumcastaneum extracts.

FIG. 2 shows the attraction of female insects to a paper impregnatedwith the sexual pheromone of the present invention.

FIGS. 3 to 10 show embodiments of the insect control device of thepresent invention as well as its individual components.

FIG. 11 shows the results of the use of the insect control device of thepresent invention in a laboratory-scale silo containing 5 kg of wheat.

FIG. 12 shows the results of the use of the insect control device of thepresent invention in a pilot-plant scale silo containing 20 kg of wheat.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described in detail below, with referenceto the figures and examples, which are included only for the purpose ofillustrating the invention and are not to be construed as limitingthereof.

The term “approximately” as used herein when referring to a measurablevalue means that it comprises variations of ±10% from the specifiedamount.

As used herein, the terms “comprises”, “has” and “includes” and theirconjugations mean “including but not limited to”.

The sexual pheromone of the present invention is biosynthesizedexclusively by male Tribolium castaneum insects and thus constitutes atrue sexual pheromone, as opposed to those of the prior art.

The insect control device of the present invention operates by a “lureand trap” mechanism. In an embodiment, insects are attracted andaggregate into the inside of the device, by crawling through theorifices of the outer shell. In an embodiment of the device, when acertain number of insects has entered the device, the resulting humidityconditions activate a bioinsecticide formulation. In the case ofbioinsecticides comprising microsclerotia, adequate humidity conditionswill cause the proliferation of conidia from the microsclerotia.

In this manner, the initial charge of bioinsecticide can be activatedseveral times and in response to the entrance and exit of insects intothe device.

Alternatively, when a single insect or a lower number of insects enterthe device, they become impregnated with a bioinsecticide in dormantform (i.e. microsclerotia). This form will yield a large number ofconidia when the humidity conditions are met, for example in theinsects' nests.

Examples

1) Extraction of Tribolium castaneum Sexual Pheromone

Insects

Specimens of pest insects of stored grains, Tribolium castaneum, wereemployed. The insects were bred in 500 mL glass jars covered with a finecloth net and kept in a breeding chamber at 27±2° C. and 70±5% relativehumidity, with a light:darkness photoperiod of 12 h/12 h.

The insects were fed a mixed diet of wheat flour (85%), brewer's yeast(5%), wheat germ (5%) and skimmed milk powder (5%).

During the pupa stage, males and females were separated taking intoaccount the morphological differences of both sexes and were kept in thediet until adult molting, thus obtaining virgin insects.

For the extraction of the pheromone of Tribolium castaneum, pools of 200male insects were employed, which were placed in individual 200 mLcontainers with 30 grs of flour and kept in the stove in theabove-mentioned conditions for a period of 1 month. Finally, the insectswere separated, and 90 grs of flour were placed in 250 mL Erlenmeyerflasks sealed with a Teflon lid with a septum to aid the extraction ofvolatile organic compounds (VOCs).

Extraction in Solid Phase

The VOCs released by Tribolium castaneum males were extracted from thehead space of the Erlenmeyer flasks containing the flour usingsolid-phase microextraction fibers (SPME) of intermediate polaritycomposed of polydimethylsiloxane/divinylbenzene and the sample was takenfor 60 minutes at room temperature (in a temperature range of 25 to 30°C.).

FIG. 1 shows the main components of the VOCs released by male and femaleTribolium castaneum, including defense pheromones (benzoquinones) andaggregate pheromones (4,8-dimethyldecanal). The detector signalcorresponding to the sexual pheromone of the present invention is onlypresent in male insects.

Isolation of the Sexual Pheromone by Capillary Gas Chromatography

The VOCs extracted by SPME were analyzed and isolated by capillary gaschromatography (CGC). A Hewlett Packard 6850 gas chromatograph with aDB-5 non-polar capillary column was used. The injector was operated insplitless mode at 250° C., He gas was used as a carrier at constant flowrate of 1.5 mL/min, the furnace temperature was programmed at 40° C. for0 min, 50° C./min at 100° C., 4° C./min at 220° C., with a retentiontime of 5 min at the final temperature. The flame ionization detector(FID) temperature was set to 280° C. This equipment is fitted with amicrocontrol valve that allowed part of the analyzed sample to bediverted to the outside of the chromatograph, which is coupled with atransfer line maintaining the temperature of the column at 220° C. Thefraction of interest was collected in a 4 mL flask at 0° C. The purityof the compound was verified using a Hewlett Packard 6890 chromatographequipped with an infernal Zebron-5HT capillary column (30 m×0.25 mmI.D., F.T. 0.25 μm, Phenomenex, USA), in the above-mentioned conditions.The CGC was operated coupled to an Agilent 5975C VL mass selectivedetector (MSD). The MSD was operated in SCAN mode with a mass range of35 to 650 u.m.a.; in electronic impact mode at 70 eV; transfer line at320° C.; ionization chamber temperature at 230° C. and quadrupole at150° C. The MSD was operated in SCAN mode with a mass range of 35 to 650u.m.a.; in electronic impact mode at 70 eV; transfer line at 320° C.;ionization chamber temperature at 230° C. and quadrupole at 150° C.

Characterization of the Sexual Pheromone

Preliminary mass spectrometry analysis revealed a molecular weight of210 g/mol, a twelve-carbon unsaturated backbone and at least onemethyl-branch.

Further analysis provided a molecular formula of C₁₄H₂₆O and a chemicalstructure having only one degree of unsaturation and a terminal carbonylgroup.

FIG. 2 shows the attraction of female insects to a paper impregnatedwith a solution comprising the extracted male pheromone.

2) Insect Control Device

FIGS. 3 to 10 show different configurations of the device of the presentinvention, representing exemplary and non-limiting embodiments of thedevice and/or individual components thereof.

The device (1) of the present invention comprises of the presentinvention an outer capsule (2) and an inner capsule (3). The capsulesare coupled to each other, and an active nucleus (4) comprising aninsecticide formulation is placed between the two capsules. The couplingof the capsules is by means of shape and are arranged concentrically. Inthis manner, the insect which is crawling in the interior of the device(1) is forced to go through the inner capsule (3) in order to reach theactive nucleus (4) comprising the insecticide.

The outer capsule (2) defines the outer shell (5) of the device, whichis provided with a plurality of orifices (6), so that insects may crawlinto or out of the device.

The inner capsule (3) may be open or closed. Open capsules allow theinsect to freely crawl about it, entering and exiting the capsuleswithout retention. Closed capsules are provided with a trap (7) at theentrance zone (8) to the active nucleus (4). This trap allows the insectto remain in contact with the inner capsule and can be achieved by meansof a conical surface (9) placed inside the inner capsule. This conicalsurface (9) allows insects crawling around the active nucleus, aided bythe roughness of the external surfaces, until they reach the conicalsurface (9). Once inside the conical surface (9), the insect will remaintrapped inside due not only to the shape and the complexity of thereverse path, but to the smooth, polished and shining surface of theconical surface (9). In this manner, insects remain in the interior ofthe inner capsule (3) until they are removed or liberated.

The capsules are provided with fit and lock mechanism (10) in theirperimeters, allowing their coupling to each other. The closing of thedevice is ensured by flexible fasteners (11), thus obtainingsemi-permanent couplings.

The device of the present invention comprises a membrane (12), locatednear the center of the interior of the device, impregnated with asubstance acting as a lure mechanism. For example, the isolated sexualpheromone of the present invention may be used. Due to its centrallocation, the membrane will generate an adequate concentration gradientof the substance in the surrounding air by diffusion and naturalconvection, thus promoting the luring effect.

Insecticides that may be used for the active nucleus comprisebioinsecticides, such as dormant forms as antipathogenic fungi, such asBeauveria bassiana microsclerotia. Since these dormant forms requirecontrolling humidity conditions in order to ensure a controlled release,their combination with the sexual pheromone of the present invention,which only attracts female insects, is advantageous to achieve acontrolled and efficient release of an insecticide formulation.

3) Use of the Insect Control Device in a Laboratory Scale Silo

FIG. 11 illustrates the attraction effect of a luring compositioncomprising the isolated sexual pheromone of the present inventionobtained from Tribolium castaneum male insects in two laboratory-scalesilo containing 5 kg of wheat of the audaz variety.

Two sets of experiments were carried out: each set consisted of a“control” silo, containing an insect control device without any luringcomposition, and an “attraction” silo, containing the insect controldevice of the present invention, i.e. with a luring compositioncomprising the isolated sexual attraction pheromone of male Triboliumcastaneum.

The left hand side of FIG. 11 shows the results for the distributions ofinsects for different positions inside the control and model siloscontaining Sitophilus sp insects. It can be seen that for the controlspecies Sitophilus sp., both the control and the attraction silos haveequivalent insect distributions.

The right hand side FIG. 11 shows the corresponding results for siloscontaining Tribolium castaneum insects. In this case, it is observedthat this species is distributed such that the number of insects closerto the insect control device is higher, which illustrates the successfuland specific attraction of Tribolium castaneum insects using the luringcomposition of the present invention.

4) Use of the Insect Control Device in a Pilot-Plant Scale Silo

The insect control device was further tested in pilot-plant scale siloscontaining 200 kg of wheat of the audaz variety.

Two experiments were carried out: the first in a “control” silo,containing six insect control devices, each one without any luringcomposition, and the second in an “attraction” silo, containing sixinsect control devices of the present invention, i.e. each one with aluring composition comprising the isolated sexual attraction pheromoneof male Tribolium castaneum.

FIG. 12 shows the results for the distributions of insects for differentpositions inside the control and model silos containing Triboliumcastaneum insects. The graph in FIG. 12 represents the percentage ofinsects found in different areas of the silo, as defined by three radialsections in the silo, corresponding to 0 cm, 15 cm and 25 measured fromthe center of the silo, as well as 8 longitudinal sections from thebottom of the silo to the loading surface.

When comparing the obtained results for the distribution of femaleTribolium castaneum insects, it can be observed that the insects areattracted to the part of the “attraction” silo containing the insectcontrol devices, as evidenced by the distribution in the right hand sideof the graph in FIG. 12, and compared to the distribution in the lefthand side of the graph in FIG. 12, corresponding to the experiment inthe “control” silo (i.e. insect control devices without any luringcomposition).

1. An isolated sexual attraction pheromone of Tribolium castaneum, saidpheromone being exclusively obtained from male insects.
 2. An isolatedsexual attraction pheromone of Tribolium castaneum wherein the pheromonehas a molecular weight of approximately 210 g/mol, a molecular formulaof C₁₄H₂₆O and a chemical structure having only one degree ofunsaturation and a terminal carbonyl group.
 3. A method to obtain anisolated sexual attraction pheromone from a plurality of male insects,comprising the steps of: placing the plurality of male insects in anairtight container comprising a solid support, placing and maintainingthe container on a stove to generate volatile organic compounds,separating the plurality of male insects from the solid support,extracting the volatile organic compounds from the solid support usingsolid-phase microextraction fibers, and isolating of the pheromone fromthe volatile organic compounds by capillary gas chromatography.
 4. Themethod according to claim 3, wherein said solid support is selected fromfilter paper and a feed medium comprising flour.
 5. The method accordingto claim 4, wherein said feed medium comprises approximately 30 g offlour.
 6. The method according to claim 3, wherein the container ismaintained on a stove for a period of approximately one month.
 7. Aninsect control device comprising: an outer shell comprising a pluralityof orifices, a central membrane impregnated with a luring composition,and an active nucleus comprising an insecticide formulation.
 8. Theinsect control device according to claim 7, wherein the luringcomposition comprises an isolated sexual attraction pheromone of maleTribolium castaneum.
 9. The insect control device according to claim 7,wherein said membrane is comprised in a capsule attached to the outershell.
 10. The insect control device according to claim 9, wherein saidcapsule comprises a conical surface.
 11. The insect control deviceaccording to claim 10, wherein said conical surface has a smooth,polished and shining surface.
 12. The insect control device according toclaim 7, wherein the insecticide formulation comprises an effectiveamount of isolated microsclerotia of Beauvaria bassiana and anagronomically acceptable carrier.
 13. The insect control deviceaccording to claim 7, wherein the active nucleus is located in a lowerportion of the device.